The Dystrophin Glycoprotein complex (DGC) is a very crucial structural component of skeletal and cardiac muscles. It is comprised of dystrophin and a plurality of proteins associated with it and imparts structural stability to the muscle membrane. The physical interactions between the proteins of the DGC form the basis for mechanical linking of the outside of the membrane to the inside and play an important role in mediating biological signaling process. These proteins form an intricate network which stabilizes the membrane as it contracts and relaxes. These interactions are essential in maintaining the structural integrity of the muscle membrane. Lack of any of these components owing to mutation compromises the structural stability leading to muscle damage.
Dystrophin was originally identified through its deficiency in the lethal neuromuscular disorder, Duchenne Muscular Dystrophy (DMD). Skeletal and cardiac muscles that lack functional full-length dystrophin protein are extremely susceptible to tear and damage from the contraction-relaxation activity. In the heart, aortic banding experiments performed on the dystrophin-deficient mdx mouse similarly result in accelerated cardiac damage. These studies demonstrated the essential role of dystrophin and the DGC in protecting the plasma membrane against contraction-induced damage.
The absence of dystrophin in DMD patients leaves the muscle membrane fragile and susceptible to damage upon contraction, leading to destruction of the DGC with loss of mechanical stability and proper mechano-transduction signaling. The dystrophin deficient myofibers undergo repeated rounds of contraction mediated injury with consequent myofiber necrosis that ultimately results in the replacement of myofibers by fibrous and fat tissue; a progressive degeneration and failure of regeneration efficiency also occurs owing to the continuous depletion of muscle precursor cells or satellite cells and their incapability to proliferate, multiply, and differentiate.
Dystrophin has four functional domains: a calponin-like actin binding domain at the amino terminal, a central rod domain of 24 spectrin-like repeats, a cysteine-rich region at the carboxy terminal, and an extreme helical carboxy terminal region. The amino terminal actin binding domain is responsible for anchoring dystrophin to cytoskeletal filamentous actin. Within the central rod domain, spectrin repeats 11 through 17 constitute a second site for binding actin. The cysteine rich region interacts with the intracellular portion of the transmembrane protein beta-dystroglycan and anchors dystrophin to the sarcolemma. The extreme carboxyl-terminal mediates its interaction with syntrophins.
The human dystrophin gene is the largest gene characterized so far. It contains 79 exons, several splicing sites and a number of tissue specific promoters that result in a range of transcripts which form amino terminal truncated dystrophin proteins of varying lengths. Dystrophin is a huge gene with an open reading frame that is 11058 nucleotides long, making it a difficult target to work with. The large size of the dystrophin gene is also responsible for its high frequency of spontaneous mutation, with most of the mutations being deletions. The extent of severity caused by these mutations varies depending on the kind of deletion. Where a deletion results in complete absence of dystrophin protein due to disruption of the reading frame of the gene, severe forms of muscular dystrophy or DMD can occur. Deletions which lead to the formation of truncated proteins result in milder forms of muscular dystrophy such as Becker Muscular Dystrophy. One deletion which removes a central part of dystrophin protein encompassing 5,106 base pairs, almost half the coding sequence, has been reported to cause a very mild form of muscular dystrophy with patients being ambulant even at the age of 61.
Dystrophin gene is one of the first genes identified by reverse genetics. DMD is an X-linked muscular dystrophy with an incidence of one in 3500 young males. DMD is one of the most common hereditary diseases known. The onset can be between the age of 3 to 5 yrs. and depending on the severity of the disease the affected males become non-ambulatory by the age of 13. The other clinical features include scoliosis, muscle weakness and damage, muscle hypertrophy, cardiomyopathy, mental retardation and very high serum creatine kinase levels. This disease ultimately causes death between the ages of 15 to 25 years. The most common cause of death in these patients is respiratory or cardiac failure. Tens of thousands of individuals are living with DMD in the United States, Europe, Australia, Canada, Israel, and Japan alone.
DMD has also been reported in a number of animals including mouse, cats and dogs. Mdx mice that have a premature stop codon mutation on exon 23 of the dystrophin gene, leading to a lack of the mature protein, have long been used as an animal model to study the pathogenesis of the disease. The absence of dystrophin results in an acute onset of skeletal muscle necrosis around 3 weeks of post-natal life, followed by an extensive period of degeneration and regeneration until necrosis gradually decreases and a relatively low level is reached in adult mice (3-4 months) with pathology stabilization. However, the pathology is far more benign than in DMD.
Vesicular Stomatitis Viruses have long been known to cause a number of diseases in humans, such as rabies. These viruses enter their hosts by making an envelope of proteins around them also known as VSV-G glycoproteins which facilitate the fusion of viral membrane with the host cell membrane. VSV-G has been widely used as a tool for gene transfer by pseudo typing viral vectors with VSV-G envelope. Recently it has been shown that LDL receptors present in the membrane of mammalian cells serve as a receptor for the VSV-G proteins and port of entry for the vesicular stomatitis viruses. This probably justifies the pantropic nature of vesicular stomatitis viruses as the LDL receptors are present in a wide range of mammalian cells and tissues.
DMD therapies that are currently being developed include DNA- and cell-based therapies, as well as drugs which aim to modulate cellular pathways or gene expression. Attempts have been made to restore the expression of full-length functional protein or short truncated protein either via exon-skipping, gene therapy, stem cells, or small molecules to induce read-through of premature stop-codon mutations. Other promising approaches include small molecules or recombinant proteins to enhance the dystrophin surrogate utrophin, and stabilize or reduce degradation of DGC.
Although the approaches previously used are promising, alternative strategies need to be developed because of the limitations of these approaches, e.g. oligonucleotides used for exon-skipping could not be effectively delivered to all the non-skeletal target muscle tissues such as heart; ataluren aimed to induce read-through of premature stop codons in dystrophin gene could only be used for a patient subpopulation exhibiting mutations displaying premature stop codons; ataluren was not potent enough to show any significant effect during clinical trials on patients treated with the drug. Currently there is no treatment available for DMD and current therapies rely in delaying the progression of the disease by clinically using Prednisone and supportive care with a mean life expectancy in the thirties.
What is desired therefore is a simple yet effective system and method for treating patients with DMD. What is also desired is a singular system and method that allows for treatment of multiple types of dystrophinopathy across a plurality of patient types, resulting in increasing or maintaining the structural integrity of the muscle fiber, limiting muscle damage, and improved muscle strength.